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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
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Neuroscience

Isolation og kultur af oculomotor, Trochlear, og spinal motor neuroner fra prænatal ISLMN: gfp Transgene mus

Published: November 12, 2019
doi:
10.3791/60440
, , , ,
1Department of Neurology,Boston Children’s Hospital, 2FM Kirby Neurobiology Center,Boston Children’s Hospital, 3Department of Neurology,Harvard Medical School, 4Medical Genetics Training Program,Harvard Medical School, 5Department of Ophthalmology,Boston Children’s Hospital, 6Department of Ophthalmology,Harvard Medical School, 7Broad Institute of M.I.T. and Harvard, 8Howard Hughes Medical Institute, 9Department of Neurology,Kokura Memorial Hospital, 10Department of Genetics,Albert Einstein College of Medicine

Summary

Dette arbejde præsenterer en protokol til at give homogene cellekulturer af primære oculomotor, trochlear, og spinal motoriske neuroner. Disse kulturer kan anvendes til komparative analyser af de morfologiske, cellulære, molekylære og elektrofysiologiske egenskaber af okulære og spinal motoriske neuroner.

Abstract

Oculomotor neuroner (CN3s) og trochlear neuroner (CN4s) udviser bemærkelsesværdig resistens over for degenerative motoriske neuron sygdomme som Amyotrofisk lateral sklerose (ALS) sammenlignet med spinal motoriske neuroner (SMNs). Evnen til at isolere og kultur primære mus CN3s, CN4s og SMNs ville give en tilgang til at studere mekanismer underliggende denne selektive sårbarhed. Til dato, de fleste protokoller bruger heterogene cellekulturer, som kan tolerere fortolkningen af eksperimentelle resultater. For at minimere problemerne i forbindelse med blandet cellepopulationer er rene kulturer uundværlige. Her beskriver den første protokol i detaljer, hvordan man effektivt renser og dyrker CN3s/CN4s sammen med SMNs-modparter fra de samme embryoner ved hjælp af embryonale dag 11,5 (E 11.5) ISLMN: gfp -transgene muse embryoner. Protokollen indeholder oplysninger om vævs dissektion og dissociation, FACS-baserede celle isolering og in vitro dyrkning af celler fra CN3/CN4 og SMN kerner. Denne protokol tilføjer en roman in vitro CN3/CN4 kultur system til eksisterende protokoller og samtidig giver en ren art-og alders matchede SMN kultur til sammenligning. Analyser, der fokuserer på motoriske neuras morfologiske, cellulære, molekylære og elektrofysiologiske egenskaber, er gennemførlige i dette kultur system. Denne protokol vil gøre det muligt at forske i de mekanismer, der definerer motorisk neuron udvikling, selektiv sårbarhed, og sygdom.

Introduction

Kulturen i primære motoriske neuroner er et kraftfuldt værktøj, der gør det muligt at studere neuronal udvikling, funktion og modtagelighed over for eksogene stressorer. Motoriske neuron kulturer er særligt nyttige for studiet af neurodegenerative sygdomme som Amyotrofisk lateral sklerose (ALS)1,2, hvissygdomsmekanismer er ufuldstændigt forstået. Interessant, på trods af den betydelige celledød af spinal motoriske neuroner (smns) i både Als patienter og Als model mus, celledød i oculomotoriske neuroner (CN3s) og trochlear neuroner (CN4s) er relativt knappe1,3,4,5,6,7,8,9. Derfor kan komparative analyser af rene kulturer af CN3s/CN4s og SMNs give vigtige fingerpeg om mekanismer, som underliggende relativ sårbarhed. Desværre, en stor barriere for sådanne analyser har været den manglende evne til at dyrke renset kulturer af disse motoriske neuroner.

Mange protokoller er blevet beskrevet for rensning af SMNs fra dyremodeller. De fleste af disse protokoller bruger tæthed gradient centrifugering10,11,12 og/eller p75NTR-antistof-baserede celle sortering panoreringteknikker 13,14,15,16. Tæthed gradient centrifugering udnytter den større størrelse af SMNs i forhold til andre rygmarvs celler, mens p75NTR er et ekstracellulært protein, der udelukkende udtrykkes af smns i rygmarven. Næsten 100% ren SMN kulturer er blevet genereret af en eller begge af disse protokoller11,12,14. Disse protokoller har dog ikke haft succes med at generere CN3/CN4-kulturer, fordi CN3s/CN4s ikke udtrykker p75NTR, og andre specifikke CN3/CN4 markører ikke er blevet identificeret. De er også mindre end SMNs og derfor vanskeligere at isolere baseret på størrelse. I stedet har in vitro-studier af CN3s eller CN4s påberåbt sig dissocieret17,18,19,20,21, explant17,22,23,24,25,26og Slice27,28 kulturer, som er sammensat af heterogene celletyper, og ingen protokoller har eksisteret for isolation og kultur af primær CN3s eller CN4s.

Her beskrives en protokol til visualisering, isolering, rensning og dyrkning af CN3s, CN4s og SMNs fra samme embryonale dag 11,5 (E 11.5) ISLMN: gfp Transgene mus29 (figur 1, figur 2a). ISLMN: gfp mærker specifikt motor neuroner med en farnesyleret gfp, der lokaliserer til cellemembranen. Denne protokol muliggør Arts-og alders matchede sammenligning af flere typer af motoriske neuroner for at belyse patologiske mekanismer i motorisk neuron sygdom.

Protocol

Alle eksperimenter, der udnytter forsøgsdyr blev udført i overensstemmelse med NIH retningslinjer for pleje og brug af forsøgsdyr og med godkendelse af Animal Care og brug Udvalget af Boston children’s Hospital. 1. opsætning af tidsindstillede parringer før dissektion At generere prænatal embryonale mus til motor neuron høst, afveje hver kvindelig mus og oprette timet parring mellem voksen ISLMN: gfp Transgene mus 11,5 dage før dagen for neuron isolation. …

Representative Results

Formålet med denne protokol var at rense og kultur både primær CN3s/CN4s og SMNs på lang sigt for at muliggøre komparative analyser af mekanismerne bag motoriske neuron lidelser (Se figur 1 og figur 2 for oversigt). Når neuronerne blev isoleret og dyrket i kultur, blev næsten ren primær CN3/CN4 og SMN-kulturer opnået (figur 5a,<str…

Discussion

Historisk set har in vitro-studier af CN3 og/eller CN4 motoriske neuroner påberåbt sig heterogene kulturer såsom adskilles17,18,19,20,21, explant17,22,23,24,25,26</su…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker Brigitte pettmann (Biogen, Cambridge, MA, USA) for instruktion i SMN dissektion teknikker; Dana Farber Cancer Institute flow cytometry Facility, den immunologiske division flow cytometry facilitet på Harvard Medical School, Joslin Diabetes Center flow cytometry Core, Brigham og Women’s Hospital flow cytometry Core, og Boston children’s Hospital flow cytometry Research facilitet til FACS isolering af primære motoriske neuroner; A.A. Nugent, A.P. Tenney, A.S. Lee, E.H. Nguyen, M.F. Rose, yderligere engle laboratorie medlemmer og projekt ALS konsortiemedlemmer til teknisk assistance og betænksomme diskussioner. Denne undersøgelse blev støttet af projekt ALS. Desuden blev R.F. finansieret af Japan Heart Foundation/Bayer Yakuhin Research tilskud i udlandet og NIH uddannelse Grant i genetik T32 GM007748; J.J. blev støttet af NIH/NEI træningsprogram i molekylære baser af øjensygdomme (5T32EY007145-16) gennem Schepens Eye Research Institute og af udviklingsmæssige Neurology Training program postdoc Fellowship (5T32NS007473-19) gennem Boston children’s Hospital; M. C. W blev støttet af NEI (5K08EY027850) og children’s Hospital Ophthalmology Foundation (fakultet Discovery Award); og E.C.E. er et Howard Hughes Medical Institute investigator.

Materials

Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) Thermo Fisher Scientific A-11001 1:400
Alexa Fluor 594-conjugated F(ab')2 goat anti-rabbit IgG (H+L) Thermo Fisher Scientific A-11072 1:400
B27 Supplement (50X), serum free Thermo Fisher Scientific 17504-044
BD FACSAria llu SORP Flow Cytometer BD Bioscience – This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers.
BD Falcon 70μm Nylon Cell Strainers CORNING 352350 For filtering the dissociating cells before FACS.
BD Falcon Round Bottom Test Tubes With Snap Cap CORNING 352054
BDNF Human ProSpec-Tany TechnoGene, Ltd. CYT-207
Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well) Greiner Bio-One International 655090 We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC
Circular Cover Glasses for microscopy Karl Hecht & Assistent 1001/14 We used this coverslip since the area was large (diamater: 14 mm).
CNTF Human ProSpec-Tany TechnoGene, Ltd. CYT-272
Cyclopiazonic acid from Penicillium cyclopium Sigma-Aldrich C1530 CPA. One of ER stressors.
4′,6-diamidino-2-phenylinodole (DAPI) Thermo Fisher Scientific D1306
Dimethyl sulfoxide Sigma-Aldrich D2650 DMSO
Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie Tips Roboz Surgical Instrument RS-5015
Forskolin Thermo Fisher Scientific BP25205
GDNF Human ProSpec-Tany TechnoGene, Ltd. CYT-305
GlutaMAX supplement Thermo Fisher Scientific 35050-061
Hanks’ Balanced Salt Solution (HBSS) Thermo Fisher Scientific 14175-095
Hibernate E BrainBits HE
Hibernate E low fluorescence BrainBits HELF Fluorescence which hinders observation of embryo's GFP expressions should be low.
Horse serum, heat inactivated, New Zealand origin Thermo Fisher Scientific 26050-070
IBMX Tocris Cookson 2845 Isobutylmethylxanthine
Laminin Thermo Fisher Scientific 23017-015
Leibovitz’s L15 medium Thermo Fisher Scientific 11415064
2-Mercaptoethanol Sigma-Aldrich M6250
Micro Dissecting Scissors Roboz Surgical Instrument RS-5913
Micro Knife 4.75" 1.7 x 27 mm blade Roboz Surgical Instrument RS-6272
Moria Mini Perforated Spoon Fine Science Tools 10370-19
mouse monoclonal antibody to neuronal class III β-tubulin (TUBB3) BioLegend 801202 1:500, TUJ1
Nikon Perfect Focus Eclipse Ti live cell fluorescence microscope and Elements software Nikon – Differential interference contrast images and immunocytochemistry images of the cell cultures were captured with these equipments
Nitric Acid 90%, Fuming (Certified ACS) Fisher Scientific A202-212 For rinsing coverslips
Olympus 1.7ml Microtubes, Clear Genesee Scientific 22-281 These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context.
Papain Dissociation System Worthington Biochemical Corp LK003150 Papain solution and alubumin-ovomucoid inhibitor solution are prepared from this kit.
Penicillin-streptomycin (10,000 U/ml) Thermo Fisher Scientific 15140-122
Phosphate buffered saline (PBS) Thermo Fisher Scientific 10010-023
Poly D-lysin (PDL) MilliporeSigma A-003-E
rabbit monoclonal antibody to Islet1 Abcam ab109517 1:200
SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 camera Nikon – Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes.
Sylgard 170 Black Silicone Encapsulant – A+B 0.9 Kg kit Dow Corning 1696157 We make dissection dishes using this kit.
TC treated Dishes, 100 x 20 mm Genesee Scientific 25-202 We make dissection dishes using this dish.
Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip Width Roboz Surgical Instrument RS-8100
Transducer for LOGOQ e VET GE Healthcare L8-18i-RS For ultrasound on female mice
Veterinary ultrasound machine GE Healthcare LOGOQ e VET For ultrasound on female mice
Zeiss LSM 700 series laser scanning confocal microscope and Zen Software Carl Zeiss – Confocal image of the embryo was captured with these equipments
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Tags

Oculomotor NeuronsTrochlear NeuronsSpinal Motor NeuronsMotor Neuron DiseaseAmyotrophic Lateral SclerosisIslet1 EGFIn Vitro CultureEmbryonic MiceMotor Neuron DevelopmentSelective Vulnerability

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Fujiki, R., Lee, J. Y., Jurgens, J. A., Whitman, M. C., Engle, E. C. Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice. J. Vis. Exp. (153), e60440, doi:10.3791/60440 (2019).
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